Gluconeogenesis and glycogenolysis in the mammalian liver are regulated in a complex manner by catecholamine and peptide hormones. Recent evidence indicates that alterations in the concentration and distribution of intracellular Ca 2 ions play important roles in mediating hormonal responses in this tissue. In particular, we've shown that treatment with norepinephrine, glucagon, or vasopressin each induces a rapid release of Ca 2 ions from rat hepatocytes. Proposed herein are studies designed to determine subcellular localization of this hormone-responsive pool of Ca 2 ions. Strong evidence suggests that chlortetracycline, a fluorescent probe of membrane-associated Ca 2 ions, is localized in the mitochondria of hepatocytes and other cells. The effect of hormonal stimuli on chlortetracycline fluorescence in intact hepatocytes will therefore be examined. In control experiments, specific alpha- and beta-adrenergic antagonists will be utilized and hormonal effects upon the fluorescence of oxytetracycline, a probe that is insensitive to cellular Ca 2 ions, will be examined. Mitochondria and microsomes will be prepared from control and hormone-treated liver tissue by standard methods. These isolated organelles will be examined for their fluorescence response, Ca content, and for the ability to accumulate Ca 2 ions during incubation in vitro. The effects of phosphorylation by purified protein kinases upon Ca 2 ions uptake by isolated microsomes will also be examined. We've shown that electron-microanalysis can monitor the Ca content of mitochondria in situ in intact spermatozoa that are transferred and examined under cryogenic conditions that preserve intracellular ionic distributions. Comparable analyses of multiple sites within individual cells from control or hormone-treated hepatocyte suspensions will indicate the extent of localization of the hormone-responsive pool of Ca. Comparison of elemental analyses thus obtained with those of control and Ca 2 ions-depleted mitochondria and microsomes may reveal characteristic features that will identify the localized areas of Ca found in intact cells. Alternatively, we will analyze thin sections, prepared by cryogenic techniques from control and hormone-treated livers, wherein mitochondria and other organelles have distinguishable electron transmission images.